Generation of antigen specific t cells

ABSTRACT

The present invention is directed to a method of generating antigen specific T cells. Furthermore, the invention is directed to antigen specific T cells, isolated transgenic TCR&#39;s, pharmaceutical compositions containing same and their use in adoptive cell therapy. This invention in particular pertains to the use of cells co-expressing allogeneic MHC molecules and antigens to induce peptide-specific T cells from non-selected allogeneic T cell repertoires.

The present invention is directed to a method of generating antigenspecific T cells. Furthermore, the invention is directed to antigenspecific T cells, isolated transgenic T cell receptors (TCRs),pharmaceutical compositions containing same and their use in adoptivecell therapy. This invention in particular pertains to the use of cellsco-expressing allogeneic MHC molecules and antigens to inducepeptide-specific T cells from non-selected allogeneic T cellrepertoires.

The adoptive transfer of lymphocytes in the setting of allogeneic stemcell transplantation (SCT) has demonstrated the power of the immunesystem for eradicating hematological malignancies (Kolb et al. 1995). Itappears that SCT can also function to eliminate solid tumors, such asrenal cell carcinomas (RCC) in some cases (reviewed in Kolb et al. 2004and Dudley and Rosenberg, 2003). In SCT recipients, the elimination ofmalignant cells may only occur after several months up to a year, due tothe fact that specific T cells must be activated in vivo and must thenexpand to adequate numbers following the development of the newhematopoietic system in the transplant recipient. Alternatively, after aperiod of time (approximately 60 days) during which tolerance isestablished in the SCT recipient, a transfer of unprimed, unseparatedlymphocytes can be made to speed up the generation of immune responsesdirected against tumor cells. Here again, the specific lymphocytescapable of attacking tumor cells must be activated and expanded from thelow frequency precursor lymphocytes that are present among theunselected population of lymphocytes that are transferred. Donorlymphocyte infusions (DLI) of unselected lymphocyte populations afterSCT work well for the elimination of chronic myelogenous leukemia (CML),which grows slowly, but are less effective in the eradication of acuteleukemia, partly due to the fact that the growth of the malignant cellsoutpaces the expansion capacity of the immune cells. This same expansiondifferential also impacts on the poor immune elimination of rapidlyprogressing solid tumors. A second handicap in the use of unselectedlymphocyte populations in DLI is that T cells may also be transferredthat have the capacity to attack normal cells and tissues of therecipient, leading to graft-versus-host-disease (GVHD), a disease withhigh morbidity and mortality.

Recent studies have demonstrated that the adoptive transfer of selectedT cells with defined peptide specificities can lead to major reductionsin tumor burden in an autologous setting, particularly if patients havebeen pretreated with non-myeloablative regimens (Dudley et al. 2002,2003). This eliminates the need to perform SCT in the tumor patient, andthereby also bypasses the problem of GVHD. Effective immune responseswere seen in pretreated melanoma patients who received autologousmixtures of tumor-infiltrating lymphocytes (TIL). These mixtures ofcells, containing both CD4 and CD8 positive T cells, appear to beclinically more efficacious than the adoptive transfer of large numbersof a single CD8 T cell clone specific for a particularMHC-tumor-associated antigen (TAA) ligand. One factor contributing tothis difference is the requirement to have CD4 T cells to maintainlong-lived CD8 T cells. Furthermore, immune responses directed againstsingle ligands can lead to the selection of tumor cell variants thathave lost expression of the corresponding ligand and thereby can escapeimmune detection. On the one hand, transfer of complex mixtures of Tcells, as they are present among tumor infiltrating lymphocytes, canovercome these problems by providing CD4 and CD8 cells with multiplespecificities but they can also lead to autoimmunity if the mixtures ofTIL contain T cells that recognize ligands expressed by normal tissues.This is demonstrated for example by the attack of normal melanocytesleading to vitiligo in melanoma patients following adoptive cell therapy(ACT) using cells that recognize melanoma differentiation antigens thatare also expressed in melanocytes (Dudley et al. 2002).

In order to extend the capacity to use ACT to treat patients with morerapidly growing tumors, it is a goal to transfer enriched,peptide-specific effector T cells (both CD4 T helper cells and cytotoxicT lymphocytes) that have been selected for their ligand specificities toeffectively attack tumor cells while avoiding serious attack of normaltissues. These cells are to be rapidly expanded to large numbers ex vivoand then used for ACT. Alternatively, the T cell receptors (TCR) of suchligand-specific T cells can be cloned and expressed as TCR-transgenes inactivated lymphocytes, using either recipient peripheral bloodlymphocytes or activated T cell clones with defined specificities thatgrow well and do not have the capacity to attack normal host tissues.

As examples, an expanded allospecific T cell clone that is specific foran MHC molecule not expressed by the recipient or an expanded T cellclone specific for a virus, such as cytomegalovirus or Epstein-Barrvirus, could be used as recipient cells for the transgenic TCR. Theavailability of a panel of transgenic TCR vectors, recognizing differentMHC-peptide ligands could be used to develop large numbers ofpre-activated T cells of both the CD4 and CD8 subtypes, thereby allowinglarge numbers of effector lymphocytes to be rapidly prepared andtransferred to patients whose tumors express the corresponding TCRligands. This would save time in achieving the numbers of specific Tcells required to control tumor growth, possibly leading to moreeffective tumor eradication of rapidly progressing tumors.

Because the determinants that specific T cells recognize on leukemia andlymphomas, as well as solid tumor cells, often represent self-peptidesderived from over-expressed proteins that are presented by self-MHCmolecules, the affinity of their T cell receptors (TCR) is low, since Tcells bearing high affinity receptors have been eliminated through theprocess of negative selection which is applied to lymphocytes duringtheir development in the thymus. More effective tumor cell recognitionoccurs if the T cells are generated from lymphocyte populations thathave not been negatively selected against self-MHC-molecules duringtheir development in the thymus.

Therefore, there is an important need to find means to rapidly generateT cells that bear TCR with high functional avidity that have thecapacity to recognize their ligands on tumor cells. Such T cells arepresent in the repertoire of an allogeneic individual who hasMHC-mismatches to the potential ACT recipient.

The work of Stauss and coworkers (Gao et al., 1999, 2000) has shown thatcytotoxic effector cells that are selected to recognize peptides derivedfrom the transcription factor, WT-1, and presented by allogeneic MHCclass I molecules have high affinity TCR and can effectively eliminateWT-1 positive leukemia without damaging normal stem cells. In thesetting of a partial-mismatched SCT, tolerance for such T cells can beestablished, allowing their specific adoptive transfer. Stauss solvedthe problem of obtaining such allo-restricted CD8 T cells by using theT2 cell line, pulsed with WT-1 peptides, as a source of stimulatingcells. T2 cells have gene deletions that impair their ability to loadendogenous peptides into their MHC class I molecules and due todeletions in chromosome 6, these cells have the limited capacity toexpress HLA-A2 molecules. When peptides are provided exogenously to T2cells they can bind to the empty HLA-A2 molecules and form stablecomplexes at the cell surface. These peptide-pulsed cells can then beused to stimulate peripheral blood lymphocytes from an HLA-A2-negativeindividual. Under such priming conditions a number of differentactivated lymphocyte populations emerge in vitro. T cells recognizingHLA-A2 molecules are activated, a fraction of which are specific for theWT-1/HLA-A2 ligand. These sought-after T cells must be separated from Tcells that recognize HLA-A2 molecules irrespective of peptide.Unfortunately, the failure of T2 cells to express a normal complement ofMHC class I molecules, as well as its inherent capacity to activatenon-MHC-restricted cells, leads to a parallel activation of NK andNK-like T cells (Falk et al 2002). These populations often dominant thecultures and it requires tedious work to eliminate these cells and toenrich the T cell populations for the desired alloreactive cells. Thisslows the process of generation of specific T cells and therefore causessignificant clinical restrictions. Furthermore, the peptides from theantigens must be known in advance since they must be pulsed onto T2cells from the outside since T2 cells lack the transporter-associatedwith antigen processing (TAP) genes which allow antigen presenting cellsto generate peptides from proteins expressed within the cell and presentthem on their surface in MHC class I and class II molecules.Furthermore, T2 only express endogenous HLA-A2 class I molecules.

US Patent Application 20020090362 discloses a method of treating apatient, the method comprising administering to the patient atherapeutically effective amount of cytotoxic T lymphocytes (CTL) whichrecognise at least part of an antigenic molecule when presented by anHLA class I (or equivalent) molecule on the surface of a cell whereinthe cytotoxic T lymphocytes are not derived from the patient. Thisapplication describes the use of stimulating cells which are allogenicor even xenogenic in view of the donor of the CTL. US Patent Application20020090362 further discloses stimulating cells which are preferablyincapable of loading a selected molecule (peptide) and in particular theuse of TAP deficient stimulating cells.

U.S. Pat. No. 6,805,861 describes a method of making a clonal populationof cytotoxic T lymphocytes (CTL) reactive against a selected moleculethe method comprising the step of (a) co-culturing a sample containingCTL derived from a healthy individual (i.e. which are not derived fromthe patient) with a stimulator cell which expresses HLA class I (orequivalent) molecules on its surface and that presents at least a partof a selected (antigenic) molecule on the surface of said stimulatorcell and (b) selecting a CTL clone reactive against said selectedmolecule when at least a part of said molecule is presented by an HLAclass I (or equivalent) molecule on the surface of a cell. Theconsiderations mentioned for US Patent Application 20020090362 alsoapply here.

Therefore, it is a problem underlying the present invention to generateT cells that bear TCR with high functional avidity that have thecapacity to recognize their MHC-peptide ligands on pathogenic agents, asfor example tumor cells. It is a further problem underlying the presentinvention to provide a method for the rapid and effective generation ofantigen specific T cells which can be used in adoptive cell transfer.Furthermore, it is a problem underlying the invention to provide a Tcell based pharmaceutical composition that can be used for treating apatient suffering from a disease without a risk ofgraft-versus-host-disease (GVHD).

The inventors have developed an alternative strategy to obtainallorestricted, peptide-specific T cells. Well known technologies toexpress proteins in dendritic cells (DCs), or other cells that canfunction as antigen-presenting cells (APCs), through the transfer of invitro transcribed RNA (Nair et al. 1998) were used. However, instead ofexpressing only RNA encoding sources of antigens, as is common in theprior art, the inventors co-transfected RNA encoding a specific targetmolecule, such as tyrosinase as a model TAA, and an RNA encoding anallogeneic MHC molecule, for example HLA-A2, into DCs, or other cells,derived from an HLA-A2 negative donor.

These transfected DC can then be used to prime PBL, for exampleautologous PBL. The HLA-A2-antigen ligands represent allo-determinantsfor the PBL of the HLA-A2 negative DC donor and therefore T cellsbearing high affinity TCR can be obtained. Because the DCs express afull complement of self-MHC molecules, the emergence ofnon-MHC-restricted populations of lymphocytes is suppressed throughnegative MHC regulation. A number of different strategies can be used toenrich the peptide-specific/HLA-A2 allorestricted T cells from T cellsthat recognize HLA-A2 independently of the specific peptide derived fromthe antigen, including cytokine capture, tetramer selection or bycloning of individual T cells that are subsequently expanded.

The present approach offers a number of advantages over the T2 system orDrosophila cells for obtaining allorestricted peptide-specific T cells(as described by Stauss and coworkers).

Firstly, T cells with allorestriction for a variety of MHC molecules canbe developed since in vitro transcribed RNA for any cloned MHC class Ior class II allele can be utilized. Thus, such APC can be used togenerate both allorestricted peptide-specific CD4 and CD8 T cells viapresentation of desired antigens through transferred allogeneic MHCclass II or allogeneic MHC class I alleles, respectively. It is notedthat the approach described in US Patent Application 20020090362 is onlysuitable for generating CD8 T cells.

Second, one is not restricted to priming against only known peptides ofselected TAA since the whole antigen is available for processing andpresentation within the DC. The antigen can be provided either in theform of protein or in the form of nucleic acid which will besubsequently used as the template to express the corresponding proteinin the APC. Also here, the differences to the prior art approaches arestriking: in US Patent Application 20020090362, the peptides are loadedonto stimulating cells (which are not professional APC's), since thestimulating cells are unable to process and present antigens. Thus, onlyknown peptides can be used in this approach and not antigens of unknownstructure as it is the case in the present invention.

Third, the TCR sequences of these selected T cells with high functionalavidity can be used for expression in autologous PBMC to generate TCRtransgenic T cells with high functional avidity for tumors that bear therespective TCR ligands. ACT can be performed using such TCR-transgenic Tcells as an alternative to the adoptive transfer of specific lymphocytesthat must be isolated and expanded over considerable periods of time invitro. This would allow treatment of patients not undergoing SCT as wellas application in patients with other non-hematological malignanciesexpressing the corresponding MHC-peptide ligands seen by the transgenicTCRs.

In particular, the present invention provides a method for thegeneration of antigen specific T cells wherein APC's are used which arederived from a healthy donor and which are autologous to PBL which arealso derived from the same healthy donor. The MHC molecule transfectedinto said APC's however is patient derived. Patient derived in thiscontext means that the sequences encoding same are directly obtainedfrom the patient or alternatively are derived from another source, forexample cDNA or genomic clones, which are identical to the MHC allelesof the patient to be treated. Furthermore, for the first time, thepresent invention uses the approach to co-transfect MHC molecule andantigen into those APC's in general in order to achieve the desiredantigen specific T cells.

The transfected APC's of the invention can be used to prime PBL whichare autologous in view of the APC's. The transferred MHC-antigen ligandsrepresent allo-determinants for the PBL of the APC donor who does notcarry the MHC gene corresponding to said MHC molecule that istransferred into the APC. Therefore T cells bearing high affinity TCRcan be obtained. Because the APCs express a full complement of self-MHCmolecules, the emergence of non-MHC-restricted populations oflymphocytes is suppressed through negative MHC regulation. The use ofAPCs, and the use of DCs in particular, in this invention is ofparticular importance because these cells can efficiently process andpresent peptides in their MHC class I and class II molecules. Inaddition, APCs are characterized by the expression of additionalcostimulatory molecules that allow them to signal additional receptorson T lymphocytes that lead to optimal activation, expansion and survivalof T cells. Furthermore, APCs and DCs in particular have the capacity tosecrete a variety of cytokines and chemokines that impinge on thefunction of the primed lymphocytes. Dependent upon the factors made bythe APCs the responding lymphocytes can be modified with respect totheir subtype, homing capacity and functional capacities. As examples,mature DCs can be used to activate T cells that have the desiredantigen-specificity due to the interactions of their TCR with theMHC-peptide ligands displayed by the DC but also have desired functionsbased on the cytokines/chemokines secreted by the particular DCs used asAPCs. DCs secreting different types of cytokines/chemokines can begenerated in vitro and used to stimulate desired types of T cells. Thus,mature DCs that are generated in culture with maturation cocktails thatlead them to secrete high amounts of the cytokine IL-12 have a goodcapacity to activate CD4 T cells of the T helper type 1 which areparticularly important in anti-tumor immunity (Napolitani et al. 2005).In contrast, immature DCs, that secrete IL-10, seem to be particularlypotent in activating regulatory T cells that can suppress the activitiesof other lymphocytes (Levings and Roncarola, 2005). Such regulatory Tcells may thereby be of clinical benefit in controlling autoagressive Tcells in patients suffering from autoimmune diseases such as type 1diabetes mellitus or in controlling immune responses to pathogens thatbecome too overriding and cause immune pathology, as for example in someforms of leprosy.

Because DCs have the capacity to process and present antigens that areexpressed in various compartments within the cell it is not necessary toknow the exact antigen peptides in order to generate T cells with thedesired antigen specificity. For example, when DCs are provided withfull protein antigens, or RNA encoding such proteins, they can processseveral different peptides from one and the same protein and presentthem in their MHC class I and II molecules; these various MHC-peptideligand can in turn prime different populations of CD4 and CD8 T cells.There is no reason why DCs can not be modified to express an allogeneicMHC class I and an allogeneic MHC class II molecule at the same time asthey are modified to express antigen, thereby allowing priming of bothCD4 and CD8 allorestricted T cells in the same cultures. Interactions ofthe activated CD4 T cells with the DCs can in turn provide them withsignals that allow them to optimally activate CD8 T cells (Toes et al.1998). DCs further can be modified to express several different antigenssimultaneously; in fact DCs can be transfected to express the entire RNAcontents of a tumor cell, encompassing many hundreds of RNA species. TheDCs are capable of processing and presenting multiple MHC class I andclass II ligands simultaneously on their surface, leading to activationof many different T cell types (Gilboa and Viewig, 2004; Geiger et al.2005; Schaft et al. 2005). There is no reason why this same property cannot be captured to create allorestricted MHC-peptide ligands byco-expressing allogeneic MHC class I and/or class II moleculessimultaneously with the mixtures of RNA or proteins for antigens.

The present invention is supported by the following experimentalresults:

-   -   1) First, the inventors showed that when RNA encoding an        allogeneic MHC molecule, such as HLA-A2, is transferred into        cells of an HLA-A2-negative donor, it can be demonstrated that        HLA-A2 molecules are expressed at the cell surface, as detected        using flow cytometry following staining with HLA-A2-specific        monoclonal antibodies. Furthermore, these cells can activate an        HLA-A2 allospecific T cell clone (JB4 cells) to secrete cytokine        (IFN-gamma), demonstrating their functional capacity. Transfer        and expression can be achieved in DCs as well as in other cells,        such as K562 cells.    -   2) Likewise, when RNA encoding a tumor-associated antigen (TAA)        such as tyrosinase as an example of a TAA for melanoma is        transferred, one can detect protein expression inside the cells,        since this is a non-membrane protein. This protein expression        can be demonstrated using flow cytometry and intracellular        staining using tyrosinase-specific antibodies and secondary        fluorescent-labeled antibodies for detection. Transfer and        expression can be achieved in DCs and in other cells such as        K562.    -   3) When one transfers both species of RNA (HLA-A2 plus        tyrosinase) into the same cells, then one can detect        simultaneous expression of both types of protein in the        recipient cells. This can be achieved in DCs and other cells        like K562.    -   4) The APCs co-expressing an MHC molecule, such as HLA-A2, and a        TAA, such as tyrosinase, generate MHC-peptide complexes and        display them on their surface in a manner with which they can        interact with T cells bearing TCR for the corresponding ligand.        This is demonstrated by the fact that such co-expressing APC can        activate a T cell clone with the specificity as measured by        cytokine release. This was demonstrated using either DCs or        other cells such as K562 as APC for an HLA-A2-tyrosinase-peptide        specific CD8 T cell clone (Tyr-F8 cells).

In particular, the present invention is directed to the followingaspects and embodiments:

According to a first aspect, the present invention provides a method ofgenerating antigen specific T cells comprising the steps of

a) providing a nucleic acid encoding a patient-derived MHC molecule andan antigen or a nucleic acid encoding said antigen;b) co-transfecting or introducing both compounds as defined in a) inantigen presenting cells (APC's) derived from a healthy donor,preferably from dendritic cells;c) priming peripheral blood lymphocytes (PBL's) derived from the healthydonor with said APC's;d) selecting those T cells which are specific for the MHC-antigenligand.

The APC's are preferably selected from dendritic cells, activated Bcells, monocytes, macrophages, activated T cells, hematologicalmalignancies with antigen presenting capacities and/or EBV-transformedlymphoblastoid cell lines.

Dendritic cells (DC) are in particular preferred. Mature dendritic cells(DCs) express both MHC class I and class II molecules at high levels,along with a wide variety of costimulatory molecules, which provide themwith the full capacity to prime naïve T cells that have not encounteredantigen previously. They also have all the necessary genes/proteins thatallow them to process and present antigens form intracellular proteinsin their MHC class I and class II molecules. Thus, they are optimalantigen presenting cells (APCs) to use as stimulating cells forinduction of both CD4 and CD8 T cells responses. Expression of RNAencoding a TAA in autologous DCs allowed tumor antigen specific T cellswith high affinity to be primed in vitro using peripheral bloodlymphocytes of the same healthy donor (Liao et al. 2004). Because DCsexpress a normal complement of self-encoded MHC class I molecules theycan negatively regulate the activity of NK and NK-like T cells, incontrast to T2 cells or cells of other species, such as Drosophilacells.

According to an embodiment, the MHC molecule and the antigen are used asa mixture of antigen and nucleic acid encoding the MHC molecule. As analternative, the nucleic acid encoding an allogeneic MHC molecule andthe antigen are provided as bicistronic RNA.

The antigens against which specific T cells should be generated arepreferably selected from pathogenic agents derived from viruses,bacteria, protozoa, and parasites as well as tumor cells or tumor cellassociated antigens, autoantigens or functional parts thereof.

The viruses are preferably selected from the group consisting ofinfluenza viruses, measles and respiratory syncytial viruses, dengueviruses, human immunodeficiency viruses, human hepatitis viruses, herpesviruses, or papilloma viruses. The protozoa may be Plasmodiumfalciparum, the bacteria tuberculosis-causing Mycobacteria.

The tumor associated antigen is preferably selected from hematologicalmalignancies or solid tumors, more preferably colon carcinoma, breastcarcinoma, prostate carcinoma, renal cell carcinoma (RCC), lungcarcinoma, sarcomas or melanoma cells.

The selection step d) is preferably performed by means of measuring thecytokine release of the T cells or other measures of T cell activation.For example, the activated T cells can be cloned as individual cells andfollowing expansion, the T cell clones can be analyzed for theirMHC-peptide specificity and those with the desired specificity can beselected for further use (Schendel et al. 1979, 1997). Alternatively,soluble MHC-peptide ligands in various forms, such as tetramers, can bemarked with a fluorescent label and incubated with the activated Tcells. Those T cells bearing TCR that interact with the tetramers canthen be detected by flow cytometry and sorted on the basis of theirfluorescence (Yee et al. 1999). Furthermore, T cells can be stimulatedfor short periods of time with tumor cells to which they should reactand their interferon gamma secretion detected by capture reagents, forexample as published (Becker et al. 2001).

According to a preferred embodiment, the method of the invention furthercomprises the step of expanding the T cells selected in d) ex vivo. Thiscan be done by co-culturing the selected T cells with APC generated inthe same manner as used for their initial priming, adding new APC to theT cell cultures every 7-10 days and providing the cells with freshculture medium on a regular basis that contains supplementary cytokines,dependent upon the type of T cell that one is expanding, this caninclude IL-2, IL-4, IL-7 and/or IL-15 among others, as described in(Schendel et al., 1997; Regn et al. 2001; Su et al. 2001, 2002).

Further, the method of the invention further comprises the step ofcloning the T cell receptor (TCR) of the isolated T cells and/orexpressing the TCR transgenes in PBMC. This can be done according toestablished methods such as those described in Engels et al., 2005.

It is one major advantage of the present approach that it is notrestricted to a specific MHC class. Thus, the MHC molecule may beselected from MHC class I, preferably HLA-A, HLA-B, HLA-C or HLA-E, orMHC class II, preferably HLA-DP, HLA-DQ, HLA-DR as long as theycorrespond to the MHC type of the patient.

According to a second aspect, the present invention provides an antigenspecific T cell, which is obtainable by the method as defined above.

Said T cell preferably is a T cell with effector cell characteristics,more preferably a cytokine producing T cell, a cytotoxic T cell orregulatory T cell, preferably CD4+ or CD8+ T cells.

In a third aspect, the invention is directed to a nucleic acid codingfor a transgenic TCR, which is obtainable by the method as explainedabove.

An additional aspect is directed to a vector, which comprises thenucleic acid coding for a transgenic TCR. This vector is preferably anexpression vector which contains a nucleic acid according to theinvention and one or more regulatory nucleic acid sequences. Preferably,this vector is a plasmid or a retroviral vector.

The invention further comprises a PBMC, which has been transformed withthe vector as defined above.

In a further aspect, the present invention provides a pharmaceuticalcomposition, which comprises the T cells or PBMCs as explained above anda pharmaceutically acceptable carrier.

Those active components of the present invention are preferably used insuch a pharmaceutical composition, in doses mixed with an acceptablecarrier or carrier material, that the disease can be treated or at leastalleviated. Such a composition can (in addition to the active componentand the carrier) include filling material, salts, buffer, stabilizers,solubilizers and other materials, which are known state of the art.

The term “pharmaceutically acceptable” defines a non-toxic material,which does not interfere with effectiveness of the biological activityof the active component. The choice of the carrier is dependent on theapplication.

The pharmaceutical composition can contain additional components whichenhance the activity of the active component or which supplement thetreatment. Such additional components and/or factors can be part of thepharmaceutical composition to achieve synergistic effects or to minimizeadverse or unwanted effects.

Techniques for the formulation or preparation and application/medicationof active components of the present invention are published in“Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa.,latest edition. An appropriate application is a parenteral application,for example intramuscular, subcutaneous, intramedular injections as wellas intrathecal, direct intraventricular, intravenous, intranodal,intraperitoneal or intratumoral injections. The intravenous injection isthe preferred treatment of a patient.

According to a preferred embodiment, the pharmaceutical composition isan infusion or an injection or a vaccine.

According to a further aspect, the present invention is directed to theuse of the antigen specific T cells or PBMCs as explained above for themanufacture of a medicament for adoptive cell therapy and in particularfor treating hematological malignancies or solid tumors and acute orchronic infections (see also above).

The present invention is illustrated by examples and figures in thefollowing.

The figures are showing the following:

FIG. 1 is showing the maturation of dendritic cells and activation ofspecific cytotoxic T cells in the conventional immune response againstmalignant cells.

FIG. 2 is illustrating the de novo priming of naive tumor-specificT-cells with RNA-pulsed dendritic cells.

FIG. 3-9 are showing the introduction of in vitro transcribed RNA.

FIG. 10 is showing the influence of RNA concentration of restimulationof TyrF8 cells by RNA-expressing DCs.

FIG. 11 illustrates the de novo priming protocol of naïve T cells withRNA-pulsed dendritic cells.

FIG. 12 shows the expression of MHC-peptide ligands on RNA-transfecteddendritic cells.

FIG. 13 depicts the tetramer staining of primed T cells fromHLA-A2-positive and HLA-A2-negative donors.

FIG. 14 shows the assessment of tetramer-sorted bulk T cell cultures forspecificity and T cell receptor avidity.

FIG. 15 depicts the assessment of T cell clones for specificity, T cellreceptor avidity and function.

EXAMPLES Description of Experiments and Figures FIG. 1. Immature andMature Stages of Dendritic Cells

Dendritic cells (DCs) are considered to be the most “professional” ofantigen-presenting cells (APC) because of their capacity to activate Tcells that have never encountered antigen (naïve T cells). This isdependent upon several factors that have already been discovered for DCsand may also be dependent upon characteristics that are still to beidentified. There are various forms of DCs and their uniquecharacteristics are continually in the process of investigation. Thetype of DC that is used most commonly for clinical studies is a myeloidDC that can be differentiated from CD14-positive blood monocytes.Through in vitro culture of such monocytes in the presence of thecytokines, granulocyte-monocyte colony stimulating factor (GM-CSF) andinterleukin-4 (IL-4), one generates an immature myeloid DC that ischaracterized by its high capacity to take up materials (includingforeign antigens) from its surroundings. When the immature DC receivesan appropriate signal during this stage it is activated to migratethrough the lymphatic vessels to the secondary lymph nodes. During thismigration and upon reaching the lymph node it undergoes further changesthat lead to a stage of full maturation. In the mature stage, the DCexpresses high levels of MHC class I and class II molecules, which allowit to present antigens in the form of peptides derived fromintracellular proteins to CD8-positive and CD4-positive T cells,respectively. The antigens that are presented by both types of MHCmolecule and displayed on the surface of DCs are generated within the DCin a complicated cellular process that includes various steps of antigenprocessing and presentation. In the case of peptides (i.e.antigen-fragments) that are presented at the DC cell surface in MHCclass I molecules it is necessary that the APC expresses moleculesencoded by the transporter-associated with antigen processing (TAP)genes, TAP1 and TAP2.

The MHC-peptide ligands that are displayed at the DC surface interactwith antigen-specific receptors present on T lymphocytes (i.e. T cellreceptors). This interaction delivers the first signal (i.e. signal 1)to the T cell; because this signal is delivered through the TCR, whichis unique for each T cell, it determines the antigen-specificity of theresultant response by selecting the T cells with fitting TCRs. MatureDCs also express a wide variety of costimulatory molecules, includingCD40, CD80, CD86 to name a few, that interact with additional receptorsexpressed by T lymphocytes and cause their further activation (i.e.signal 2). Furthermore, DCs have the capacity to secrete a variety ofsoluble molecules, including cytokines and chemokines, which can attractdifferent subtypes of lymphocytes to their vicinity and can impinge onthe differentiation of the T cells receiving signal 1 and signal 2.Following binding to a further set of receptors expressed by lymphocytesthese factors can deliver yet another signal to the lymphocytes whichcan influence their differentiation and ultimate function. This issometimes referred to as signal 3.

It is possible to generate immature and mature DC in vitro. For examplemonocytes can be obtained by various procedures (positive selection ofCD14-positive cells using commercial kits for cell selection, by theirproperty of adherence to plasticware, or by their size and density usingthe process of cell elutriation). These monocytes are then cultured withGM-CSF and IL-4 to yield populations of immature DCs. Differentcombinations of reagents can be used to induce maturation of DCs and inparallel to alter their secretion of soluble mediators. These differentforms of immature/mature DCs in turn can be used to activate T cellsthat not only have the desired antigen-specificity due to theinteractions of their TCR with the MHC-peptide ligands displayed by theDC but also having desired functions based on the cytokines secreted bythe DCs. Thus, mature DCs that are generated to secrete high amounts ofthe cytokine IL-12 have a good capacity to activate CD4 T cells of the Thelper type 1 which are particularly important in anti-tumor immunity.

If properly activated and selected, T cells have the capacity torecognize and destroy tumor cells or cells infected with variouspathogens. In the case of tumor cells, the tumor-associated antigens(TAA) that are processed and presented by APCs often represent moleculesthat are self-proteins that are overexpressed in tumor cells. Becausemost T cells that bear TCR that interact with MHC-peptide ligandsderived from self molecules are eliminated in the thymus in a processknown as “negative selection” there are few T cells in the repertoirethat have TCR with sufficient avidity to recognize tumor cellsexpressing such TAA-ligands.

FIG. 2. De Novo Priming of NaïVe Tumor-Specific T Cells with RNA-PulsedDendritic Cells

In order to obtain T cells that have better capacity to recognizeMHC-peptide ligands on tumor cells or other cells, such as thoseinfected with pathogens, one can tap a non-selected repertoire of Tcells from a healthy individual by presenting peptides derived from theselected antigen/antigens via allogeneic MHC molecules. For example, Tcells from a healthy donor A, who is not HLA-A2, have not been exposedto HLA-A2-peptide complexes in the thymus, therefore there are T cellsavailable among the peripheral blood mononuclear cells (PBMC) of such anindividual that express TCR capable of interacting with HLA-A2-peptidecomplexes with high avidity. In order to stimulate naïve T cells bearingsuch desired TCR, DC of said donor A are generated from monocytes andare then modified to express an allogeneic MHC class I or class IImolecule. Here an example is provided for HLA-A2. This molecule is anallogeneic MHC molecule for T cells of donor A, who is not HLA-A2. Byintroducing HLA-A2 encoding RNA into DCs of donor A, it is possible tocreate a population of DCs that express HLA-A2 molecules at their cellsurface. Hereby, the DC still expresses its normal set of self-MHCmolecules that are encoded by the chromosomal MHC genes of donor A, butin addition it expresses the allogeneic MHC molecule which is translatedfrom the introduced RNA. At the same time the DC can be provided withRNA encoding a protein antigen from which peptides can be processed bythe DC and presented at the cell surface by the allogeneic HLA-A2molecule. When DCs expressing such allogeneic MHC-peptide ligands attheir cell surface are used as APC to stimulate naïve T cells they canactivate T cells with TCR that interact with the desired allogeneicMHC-peptide ligands. Not all T cells recognizing the allogeneic MHCmolecules will have the desired peptide specificity, therefore the cellswith the desired TCR must be selected from the full population ofactivated T cells. There are several procedures which are well known toimmunologists to achieve this selection. For example, the activated Tcells can be cloned as individual cells and following expansion, the Tcell clones can be analyzed for their MHC-peptide specificity and thosewith the desired specificity can be selected for further use.Alternatively, soluble MHC-peptide ligands in various forms, such astetramers, can be marked with a fluorescent label and incubated with theactivated T cells. Those T cells bearing TCR that interact with thetetramers can then be detected by flow cytometry and sorted on the basisof their fluorescence.

The T cells that are selected with the desired TCR specificity may thenbe expanded in vitro for use in adoptive cell therapies (ACT) ofpatients whose tumors or infected cells express the correspondingHLA-A2-peptide ligands. Alternatively, the TCR sequences can be isolatedand determined for such T cells and used to generate TCR constructs thatcan be introduced into lymphocytes or other cells to generate transgenicTCR cells that can be used for ACT.

FIG. 3. Introduction of In Vitro Transcribed RNA into K562 Cells

As a first experimental approach to demonstrate the feasibility of thisstrategy we used the K562 cell line as a recipient cell line forexpression of an allogeneic HLA-A2 MHC molecule following RNA transfer.This cell line is derived from an erythromyeloid leukaemia and is wellknown to cellular immunologists. Because it is a tumor line it can begrown to large numbers and is easy to handle experimentally.Furthermore, it does not express any MHC class I molecules on its cellsurface therefore expression of allogeneic MHC molecules can be easilydetected at the cell surface of K562, following introduction of nucleicacids encoding MHC class I molecules.

In the experiments summarized in this figure, HLA-A2 mRNA, which wasgenerated by in vitro transcription from an HLA-A2-encoding cDNA, wasintroduced into K562 cells via electroporation. The transientlyRNA-transfected cells were compared with a line of K562 cells that werestabily transfected with the gene encoding HLA-A2, designated as K562-A2cells.

The surface expression of HLA-A2 molecules was determined by flowcytometry following incubation of the cells with a monoclonal antibodyspecific for HLA-A2 molecules and the data are presented as percentagesof positive cells (left hand figure). As a mock control the K562 cellswere electroporated in a cuvette containing water instead of HLA-A2mRNA. At all time points subsequent to electroporation, no positivecells were detected in the mock control. Three different amounts ofHLA-A2 RNA were used for electroporation of the same number of K562cells. The highest percentage of HLA-A2 positive cells was detectedusing the amount of 24 micrograms of RNA and the highest percentage ofcells was seen at 11 h after electroporation. The percentages ofpositive cells expressing HLA-A2 molecules following electroporation ofRNA were comparable at 11 h and 24 h to those of the positive controlK562-A2 cells that stabily express the HLA-A2 gene. The transientexpression of HLA-A2 following introduction of HLA-A2 mRNA isdemonstrated by the decrease in percentages of positive cells at 48 hand no detection at 120 h.

To determine whether T cells bearing TCR that recognize HLA-A2 moleculesas an alloantigen could be activated by HLA-A2 RNA-expressing cells, a Tcell clone designated as JB4 was studied. This T cell clone secretes thecytokine interferon-gamma when its TCR interacts with HLA-A2-peptideligands. The exact peptide/s required for this recognition is not knownbut is expressed in K562 cells and in DCs and in most other cells thatwe have tested, thus it appears to be derived from a ubiquitous protein.When JB4 cells are cocultured with K562 mock transfected cells, noIFN-gamma release is detected (right hand figure). Strong release isseen with the positive control of K562-A2 cells. All K562 cellstransfected with various amounts of HLA-A2 RNA could induce secretion ofcytokine by JB4 cells, albeit at lower levels than the positive control.Nevertheless, these are substantial levels of cytokine release. Incultures of K562 or DCs that did not have addition of JB4 cells, nocytokine release was measured, demonstrating that the stimulating cellsdo not secrete interferon-gamma themselves. A final control of JB4 cellswithout stimulating cells showed no cytokine release and demonstratedthat the T cells must be activated by the appropriate MHC-peptide ligandto secrete interferon-gamma.

These studies showed that introduction of an allogeneic MHC moleculeinto K₆₅₂ cells via transient RNA expression led to cell surfaceexpression of HLA-A2 molecules and the cells could activate an HLA-A2alloantigen-specific T cell clone.

FIG. 4. Introduction of In Vitro Transcribed RNA into DCs.

A similar experimental approach was used as described in FIG. 3 toassess surface expression of HLA-A2 molecules on DCs followingelectroporation of HLA-A2 mRNA and to assess their capacity to stimulatecytokine secretion from JB4 cells. Here an amount of 24 micorgrams ofHLA-A2 mRNA was used throughout but three different sources of mRNA werecompared. These included a commercial source of in vitro transcribed RNAin which the 3′ UTR of the human alpha-globulin gene was added to theHLA-A2 gene construct, with the intention to stabilize the RNAexpression inside the DCs. This RNA was compared to polyadenylatedHLA-A2 RNA generated using a commercial procedure and kit from Ambionversus a similar RNA generated using a commercial procedure and kit fromCureVac. The polyadenylated mRNA generated using the Ambion procedureand introduced by electroporation into DCs cultured from an HLA-A2negative donor showed the best results. The highest percentages ofHLA-A2 positive cells were found with this mRNA, the appearance ofHLA-A2 molecules at the surface of the DCs was detected earlier (at 6 h)and expression in the DCs was still found at 120 h using this source ofmRNA. All further experiments therefore utilized this source of mRNA.

The DCs were studied for their capacity to stimulate cytokine secretionfrom JB4 cells as described in FIG. 3. Mock control DCs (most leftcolumn) induced only background levels of cytokine release. Higherlevels were seen using DCs expressing the three different sources ofmRNA but those DCs electroporated with Ambion mRNA were superior. The DCcultures containing no JB4 cells did not have interferon-gamma,demonstrating that it is only released by the JB4 cells followingstimulation with HLA-A2-RNA expressing DCs.

FIG. 5. Introduction of In Vitro Transcribed RNA Encoding theTumor-Associated Antigen Tyrosinase into K562-A2 Cells.

RNA was generated using the Ambion procedure and specific for theantigen tyrosinase. It was electroporated into K562-A2 cells and surfaceexpression of HLA-A2 was measured using a monoclonal antibody specificfor HLA-A2 molecules. The green curves represent staining with anisotype control antibody and the blue curves staining with theHLA-A2-specific antibody. Here HLA-A2 surface expression was found atall time points since K562-A2 cells constitutively express the HLA-A2gene. A decrease in expression of HLA-A2 was noted at 12 h which may berelated to a stress-induced impact as a result of electroporation. Thecells recovered high expression of HLA-A2 molecules thereafter. Proteinexpression of tyrosinase was measured following intracellular detectionof protein using a monoclonal antibody specific for tyrosinase. Theorange curves represent the staining of the mock control and the bluecurves represent the tyrosinase protein inside the cells. A slight shiftin staining could be seen at several time points indicating that proteinwas being made following introduction of the tyrosinase RNA.

FIG. 6. Expression and Function of Tyrosinase in K562-A2 Cells

Tyrosinase RNA was electroporated into K562-A2 cells and the percentagesof positive cells as well as the intensity of staining, designated asmean fluorescent intensity (MFI), was determined at various time points(upper left field). The red columns show the constitutive HLA-A2expression on K562-A2 cells, again with a drop in level between 11 and24 h and recovery thereafter. The blue columns show the intracellularstaining of tyrosinase protein with the peak at 6 h. The shift instaining at 6 h is illustrated in the lower right field in which themock control staining is shown by the orange curve and the tyrosinasestaining shown by the blue curve. The fold increase in expressioncompared to the mock control, over time, is shown in the lower leftfield, giving a maximum at 11 h. The interferon-gamma secretion of a Tcell clone, Tyr-F8, which sees a tyrosinase-derived peptide presented byHLA-A2 molecules is shown in the upper right field. K562-A2 cells do notinduce interferon-gamma secretion by Tyr-F8 T cells, but they do sofollowing electroporation with tryosinase RNA. The T cells alone do notsecrete cytokine but they can also be stimulated by K562-A2 cells loadedwith synthetic peptide from tyrosinase or by melanoma cells thatco-express tyrosinase and HLA-A2 (MEL-IL2 cells).

This demonstrates that introduction of RNA for tyrosinase into K562-A2cells allows them to process and present the appropriate MHC-peptideligand that is seen by the TCR of Tyr-F8 cells and activates the T cellsto secrete interferon-gamma. RNA transfected cells used 12 and 24 hafter electroporation had comparable stimulatory capacities.

FIG. 7. Introduction of In Vitro Transcribed Tyrosinase RNA in DCsGenerated from HLA-A2 Positive Donors.

A similar set of experiments was done as those described in FIG. 6except DCs from an HLA-A2-positive donor were used instead of K562-A2cells. Expression of intracellular tyrosinase protein was detected inthe DCs at various time points following electroporation of RNA (upperpanels). The highest mean fold expression over the mock control was seenat 3 h in the DCs. DCs alone could not stimulate cytokine secretion byTyrF8 cells but they could do so following electroporation withtyrosinase RNA. DCs used either 12 h or 24 h after electroporation hadthe capacity to activate Tyr-F8 T cells to secrete interferon-gamma.

FIG. 8. Co-Expression of HLA-A2 and Tyrosinase in K562 Cells.

K562 cells which do not express any HLA molecules were electroporatedwith RNA for HLA-A2 and tyrosinase. Either each species of RNA wasintroduced individually or both in combination. The upper right panelsummarizes the protein expression of HLA-A2 alone at different timepoints (most left set of columns) tyrosinase alone, followed by HLA-A2staining in cells electroporated with both HLA-A2 and tyosinase RNA andthe tyrosinase staining in cells receiving both species of RNA. Optimalexpression of both proteins in cells electroporated with both species ofRNA was seen at 24 h. The K562 cells co-expressing both RNAs couldstimulate interferon-gamma secretion from TyrF8 cells 12 h and 24 hafter electroporation.

FIG. 9. Co-Expression of HLA-A2 and Tyrosinase in DCs Made from HLA-A2Negative Donors.

Co-expression of HLA-A2 protein and tyrosinase protein in DCs preparedfrom an HLA-A2 negative donor was analyzed as described in FIG. 8.Co-expression of both proteins was found in DCs at various time points.The levels of HLA-A2 expression seemed to be reduced in the presence oftyrosinase RNA. Nevertheless the DCs co-expressing both RNAs couldstimulate significant levels of interferon-gamma by TyrF8 cells abovethose of the T cells alone or following incubation with DCselectroporated with the single species of RNA.

FIG. 10. Influence of RNA Concentration of Restimulation of TyrF8 Cellsby RNA-Expressing DCs.

The capacity of DCs to stimulate cytokine secretion from TyrF8 cells wasanalyzed using DCs prepared from an HLA-A2 negative donor transfectedwith different amounts of HLA-A2 and tyrosinase RNA. Mock control DCs(most left column) induced only background levels of cytokine release.The T cells alone do not secrete cytokine but they can also bestimulated by melanoma cells that co-express tyrosinase and HLA-A2(MEL-IL2 cells).

All DCs transfected with various amounts of both RNAs could inducesecretion of interferon-gamma by TyrF8 cells, whereas those DCselectroporated with 24 μg HLA-A2 and 24 μg tyrosinase RNA or 48 μg RNAHLA-A2 and 24 μg tyrosinase RNA were superior indicating that less RNAfor the restimulation of T cells may be better.

FIG. 11. De Novo Priming Protocol of NaïVe T Cells with RNA-PulsedDendritic Cells

In order to analyze the capacity of RNA-loaded DCs to prime naïve Tcells to become tumor-specific lymphocytes, a three phase primingprotocol was developed. Eight days in advance of the initiation ofpriming (day −8), monocytes were prepared by plastic adherence from anHLA-A2-positive donor and an HLA-A2-negative donor, as described. Thesepurified monocyte populations were used to prepare immature DCs thatwere then matured as described. On the first day (day 0) of the T cellpriming protocol the DCs were loaded with RNA via electroporation, asdescribed. The DCs derived from the HLA-A2-positive donor were loadedwith 24 μg of tyrosinase RNA as a model tumor antigen. Tyrosinaseprotein translated from the RNA in the DCs should be processed andpresented as peptides in association with the self-encoded HLA-A2molecules at the DC surface. The DCs derived from the HLA-A2-negativedonor were loaded with RNA encoding HLA-A2 (48 μg) and tyrosinase (24μg). Here the HLA-A2 molecules encoded by the transferred RNA representallogeneic MHC molecules. Peptides derived from the tyrosinase proteinshould be processed and presented by HLA-A2 molecules that aretranslated from the HLA-A2 encoding RNA transferred to the DCs; thesepeptide-MHC complexes represent allorestricted-peptide ligands. On day 0of the priming protocol, autologous CD8⁺ T lymphocytes from each donorwere isolated via negative selection using a commercial kit and themanufacturer's instructions (CD8⁺ T cell Isolation Kit II (human),Miltenyi, Bergisch Gladbach, Germany) to >80% purity. These untouchedautologous CD8⁺ T cells were added to the mature RNA-pulsed DCs 9 hafter electroporation at a ratio of 10:1 in AimV medium (Gibco BRL,Karlsruhe, Germany) containing IL-7 (10 ng/mL). IL-2 (20 IU/mL) wasadded after 2 days and then on every 3^(rd) subsequent day. Seven days(day 7) after the 1^(st) stimulation, the 2^(nd) stimulation of thepriming cultures was performed using RNA-pulsed DCs prepared in the samemanner as for the 1^(st) stimulation and using the same cultureconditions as used on day 0 of priming. After an additional 7 days (day14), HLA-A2-restricted tyrosinase-specific T cells were sorted with theaid of a phycoerythrin (PE)-labeled HLA-A*0201/htyr369-377peptide/humanβ₂m tetramer (Wölfl et al., 2004; provided by Prof. D. Busch, Instituteof Medical Microbiology, Immunology and Hygiene, Technical University,Munich, Germany). Some of the positively-selected T cells were plated at1 cell/well in 96-well V-bottomed plates (TPP, Trasadingen,Switzerland). The wells were fed with Aim V medium containing 50 IU/mLIL-2 (Chiron Behring, Marburg, Germany) every three days and 5 ng/mLIL-7 (Promokine, Heidelberg, Germany) and 10 ng/mL IL-15 (PeproTechInc., New Jersey, United States) every seven days. Selected clones andthe remaining tetramer-selected uncloned T cells were maintained inculture in Aim V medium, with IL-2, IL-7, IL-15, as above, and wereprovided with feeder cells, comprised of peripheral blood mononuclearcells derived from a pool of five donors which was irradiated with 50Gy. The T cells were stimulated non-specifically with anti-CD3 antibody(0.1 μg/mL; provided by Dr. Elisabeth Kremmer, Institute of MolecularImmunology, GSF, Munich, Germany) every two weeks.

FIG. 12. Expression of MHC-Peptide Ligands on RNA-Transfected DendriticCells

To demonstrate that the DCs used for T cell priming expressed theexpected MHC-peptide ligands, the expression of HLA-A2 molecules at thecell surface and intracellular tyrosinase protein was determined usingflow cytometry as described previously. Co-expression of HLA-A2molecules and tyrosinase protein was detected at high levels in DCsderived from both donors. The levels of intracellular tyrosinase proteinwere comparable in the DCs prepared from both the HLA-A2-positive andthe HLA-A2-negative donor. The levels of HLA-A2 surface expression on amajority of cells were higher in the DCs prepared from theHLA-A2-positive donor where they are encoded by an endogenous gene.Whereas the levels of transgenic HLA-A2 molecules are more variable onthe DCs prepared from the HLA-A2-negative donor.

The capacity of such DCs, using cells prepared according to the sameprotocol in independent experiments, to stimulate T cells was determinedusing an HLA-A2-alloresponsive T cell clone that recognizes HLA-A2molecules irrespective of the peptides they carry (i.e. HLA-A2+xyzpeptides). Both populations of DCs were able to stimulate this T cellclone (JB4) to secrete interferon-gamma, as measured in a standard ELISAas described previously. No cytokine release was seen from T cellsincubated without DCs. The presence of ligands comprised of HLA-A2molecules presenting a tyrosinase-derived peptide, was assessed bymeasuring interferon-gamma release by a T cell clone (Tyr-F8) specificfor HLA-A2-tyrosinase peptide. T cells incubated without DCs releasedonly background levels of cytokine. In this experiment theHLA-A2-positive DCs were provided with 48 μg of tyrosinase whereas theHLA-A2-negative DCs were provided with 24 μg of tyrosinase.

These results demonstrated that the transgenic HLA-A2 molecules,provided by RNA transfer into the DCs derived from the HLA-A2-negativedonor, could be stained at the cell surface with specific HLA-A2antibody and recognized by an HLA-allospecific T cell clone (JB4). Thetransfer of tyrosinase RNA led to intracellular protein expression inboth DC populations. The co-transfer of HLA-A2 and tyrosinase RNAallowed an HLA-A2-tyrosinase specific T cell clone (Tyr F8) to beactivated, as did the transfer of tyrosinase RNA into DCs expressingendogenously encoded HLA-A2 molecules.

FIG. 13. Tetramer Staining of Primed T Cells from HLA-A2-Positive andHLA-A2-Negative Donors

To select T cells bearing T cell receptors specific forHLA-A2-tyrosinase peptide ligands that are present among the primed Tcell cultures, the T cells were stained with anHLA-A*0201/htyr369-377/Hβ₂m tetramer and analyzed by flow cytometry.Cells from each of the two different priming cultures that showedtetramer binding were gated (black rectangle, n) and isolated in aMoFlo™ High-Performance Cell Sorter (Dako, Fort Collins, Colo., USA).The CD8+tetramer⁺ T cell population of the HLA-A2-positive donor showedtetramer binding at only a low intensity whereas the CD8⁺tetramer⁺ cellsfrom the HLA-A2-negative donor were stained at a higher intensity. Thesorted tetramer⁺ T cells were plated as single cell and the remainingcells retained in culture as uncloned bulk cultures.

These results demonstrated that the priming protocol yielded T cellsthat carried TCR capable of binding HLA-A2-tyrosinase-specifictetramers. More T cells bound the tetramer at higher intensity in thecultures primed using the DCs derived from the HLA-A2-negative donor,indicating better stimulation by HLA-A2 allogeneic, peptide ligands.

FIG. 14. Assessment of Tetramer-Sorted Bulk T Cell Cultures forSpecificity and T Cell Receptor Avidity

The sorted cells that were retained as bulk cultures were reanalyzed fortetramer staining after 8 days. As expected, the majority of sortedcells were CD8⁺ in the sorted bulk cultures derived from both of thepriming cultures. Tetramer⁺ T cells were present at a frequency of about8% in the cultures primed using HLA-A2-positive DCs. These cells had anintermediate intensity of tetramer binding (MFI=127.49), whereas 80% ofthe bulk culture T cells from the HLA-A2-negative donor bound thetetramer with a much higher intensity (MFI=3248.77). The intensity oftetramer binding is one parameter used to assess T cell receptor (TCR)avidity for a particular MHC-peptide ligand (Yee et al., 1999). A higherintensity of tetramer binding indicates a better interaction of theligand with the TCR. A second parameter of TCR avidity is the tetrameroff-rate (Palermo et al., 2005). T cells are incubated with tetramer andare then washed and incubated in the absence of tetramer and thepresence of antibody specific for HLA-A2 molecules. The presence of thisantibody prevents tetramers which have fallen off the surface of the Tcells from rebinding to the cell surface. The intensity of tetramerstaining on the T cells is determined at various time points afterwashing: 0 h, 1 h, 2 h. If the tetramer staining is rapidly lost, thisindicates that the TCR has only low avidity for the MHC-peptide ligandof the tetramer. If tetramer binding is more stable over time, thisindicates a higher TCR avidity for the MHC-peptide ligand. In thecomparison of the two bulk cultures, it was found that only 20% of theoriginal tetramer-binding cells (1.7% vs 8.3%) were still tetramer⁺after 2 h in the cells derived from the HLA-A2-positive donor. Incontrast, 75% of the cells were still tetramer⁺ after 2 h in the T cellsderived from the HLA-A2-negative donor (60.5% vs 80.6%).

The specificity of tetramer binding was determined using a tetramercomprised of HLA-B7 molecules presenting a peptide derived fromcytomegalovirus (B7-CMV tetramer, provided by Prof. D. Busch). Both bulkT cell cultures showed only low levels of staining (0.3-0.4%). Inaddition, an HLA-A2-CMV peptide tetramer was also used. Only 0.4% of Tcells from the HLA-A2-positive donor bound this tetramer, whereas 12.7%of the bulk T cells from the second donor were positive. These T cellslikely represent T cells with TCR that recognize HLA-A2 molecules asalloantigens, irrespective of the peptides they present. The intensityof tetramer binding was lower (MFI=166.98) compared to the intensity ofthe HLA-A2-tyrosinase-tetramer binding (MFI=3248.77).

These results demonstrate that T cells bearing TCR with higher avidityas defined by intensity of tetramer staining and slower tetrameroff-rate were derived from the cultures using HLA-A2-negative DCs,demonstrating the superiority of inducing responses in non-negativelyselected T cell repertoires, using allogeneic-peptide ligands.

FIG. 15. Assessment of T Cell Clones for Specificity, T Cell ReceptorAvidity and Function

From the single cell plating experiments, two clones were selected forfurther characterization. One clone (PS P4D11) was derived from theHLA-A2-positive donor and one clone (SW P12B10) was derived from theHLA-A2-negative donor.

The PS P4D11 clone, from the HLA-A2-positive donor, showed anintermediate intensity of tetramer staining (MFI=166.98) with a rapidrelease of tetramer as measured at 1 h and 2 h. This T cell clone couldnot be stimulated to secrete interferon-gamma following incubation withT2 cells loaded with specific peptide (T2 cells provided by PeterCresswell, Yale University, New Haven, Conn.) nor following stimulationwith tumor cells, expressing HLA-A2 and tyrosinase (MeI 624.38 and MeIIL-2). It could, however kill HLA-A2⁺tyrosinase⁺ melanoma tumor cellsbut not HLA-A2⁺tyrosinase⁻ tumor cells, demonstrating its specificityfor HLA-A2-tyrosinase ligands.

The SW P12B10 clone, derived from the HLA-A2-negative donor, showed ahigher intensity of tetramer binding (MFI=1009.04) and the stability oftetramer binding was higher with all of the cells still positive fortetramer after 2 h. This clone showed specific interferon-gamma releasefollowing stimulation with T2 cells loaded with tyrosinase-derivedpeptide, but not against T2 cells without peptide, demonstrating peptidespecificity. It also could be stimulated to release interferon-gammafollowing stimulation with HLA-A2⁺tyrosinase⁺ melanoma tumor cells, butnot against HLA-A2-negative, tyrosinase positive melanoma cells (SK MeI28) or against HLA-A2-positive, tyrosinase negative melanoma tumor cells(MeI A375). This pattern demonstrated that the clone was specific for anHLA-A2-tyrosinase peptide. This clone could also kill HLA-A2-positive,tyrosinase positive melanoma tumor cells, but not HLA-A2-positive,tyrosinase negative tumor cells.

These results show that the T cell clones derived from the two differentpriming cultures showed differences with respect to TCR avidity, asdetected by intensity of tetramer binding and tetramer off-rate, withthe allorestricted peptide-specific T cell clone having TCR of higheravidity by this definition. Furthermore, this T cell clone showedfunctional superiority since it could not only kill melanoma cellsspecifically, but could also be activated to secrete interferon-gammafollowing peptide stimulation and following stimulation with tumorcells, in an HLA-A2, tyrosinase-specific manner.

Materials and Methods

Adherent tumour cell lines, MeI A375, MeI IL-2, MeI 624.38 and SK MeI 28

These human melanoma cell lines were cultured in RPMI 1640 mediumsupplemented with 12% fetal bovine serum, 1× Non-essential amino acids,2 mM L-glutamine and 1 mM sodium-pyruvate. Volume of the medium in amiddle-sized culture flask was 10 mL. Approximately every 3-4 days cellsgrew to confluence. Depending on the growth rate of individual celllines they were split 1:2 to 1:10. The medium was removed, cells werewashed once with PBS and then incubated with 2 mL trypsin/EDTA 2× for 3min at room temperature. Detached cells were resuspended in fresh RPMI1640 medium plus supplements.

T cell clones, JB4 and Tyr-F8

T cell clones were first expanded in standard restimulation cultures.Large stocks were aliquoted and frozen. Each time they were needed foran experiment, T cells were taken from the stock, thawed, placed intowells of a 24-well plate and restimulated according to standardisedprotocols.

Suspension tumour cell lines, T2, K562 and K562-A2 Cells

T2, K562 and K562-A2 were cultured in RPMI 1640 medium supplemented with12% FBS, 1×MEM, 2 mM L-glutamine and 1 mM sodium-pyruvate. Volume of themedium was 10 mL in a middle-sized culture flask. Approximately every 4days, ¾ of the cell suspension was removed and the same volume of freshmedium was added. For the K562-A2 cells, the medium was supplementedwith 1 mg/mL of the selection antibiotic G418.

DC Generation and Culture

PBMCs from a donor were isolated by Ficoll density gradientcentrifugation. PBMCs were resuspended in RPMI 1640 medium supplementedwith 1% autologous plasma at 7.5×10⁶ cells per T75 culture flask. Theflasks were incubated at 37° C. and 5% CO₂ for 1 hr. Non-adherent cellswere carefully washed away. Adhering monocytes were cultured in 10 mLper flask of dendritic cell medium supplemented with 1% autologousplasma, 800 U/mL GM-CSF and 800 U/mL IL-4. After 3 days of culture, 800U/mL GM-CSF and 800 U/mL IL-4 were added again. On day 6 of culture, 800U/mL GM-CSF, 500 U/mL IL-4, 5 ng/mL IL-1β, 9 ng/mL IL-6, 9 ng/mL TNF-αand 2 μM PGE₂ per 1 mL were added to the immature DCs to inducematuration. Mature DCs were harvested on day 8 of culture.

Flow Cytometry

Flow cytometry was used to measure the cellular expression of chosenmolecules. These molecules are specifically stained with monoclonalantibodies attached to fluorescent dyes. Flow cytometric analysis wasperformed in the fluorescence-activated cell sorter (FACS™). In theFACSCalibur™, the suspension of stained cells was forced through anozzle, creating a fine stream of liquid containing cells spaced singlyat intervals. As each cell passed through a laser beam, it scattered thelaser light and fluorescent molecules associated with the cell wereexcited. Sensitive photomultiplier tubes detected both the scatteredlight, which gave information about the size and granularity of thecell, and the fluorescence emissions, which gave information about thebinding of the labelled monoclonal antibodies and, hence, the expressionof targeted molecules by each cell. For sorting of the cells ofinterest, a MoFlo™ High-Performance Cell Sorter (Dako, Fort Collins,Colo., USA) was used with 25,000 events/s and max. 1 psi.

Direct Staining of Cell-Surface Molecules

In order to measure the expression of HLA-A2 molecules on the surface,the hybridoma supernatant HB82 (ATCC, Bethesda Md.) and a secondary goatanti-mouse antibody conjugated with PE (phycoerythrin) were used for thedirect staining on the cell surface. Approximately 1×10⁵ DCs, K562 orK562-A2 cells were washed once. Each washing step included resuspensionof cells in 600 μL of FACS™ buffer, centrifugation at 300×g for 4 minand discarding of the supernatant. After washing, 50 μL of HB82 wasadded separately to the pellets. After a 30 min incubation period onice, cells were washed once. Then 0.5 μL of the secondary antibody (1/100 dilution; 0.5 μL antibody in 50 μL FACS™ buffer) were addedseparately to the pellets. After a 30 min incubation period on ice andin the dark, cells were washed once. Finally, cells were resuspended in200 μL of FACS™ buffer and flow cytometric analysis was performed.

Indirect Staining of Intracellular Molecules

In order to measure the expression of tyrosinase inside RNA-transfectedDCs, cells were first fixed using 1% paraformaldehyde in FACS™ buffer.After the fixation the cells were permeabilised using 0.1% and 0.25%Saponin in FACS™ buffer. Briefly, approximately 3×10⁵ cells were washedonce. Each washing step included resuspension of cells in 500 μL ofFACS™ buffer or 0.1% Saponin in FACS™ buffer, centrifugation at 300×gfor 4 min and discarding of supernatant. After washing, 500 μL of 1%paraformaldehyde in FACS™ buffer (fixation medium) were added to thepellet and mixed gently. After a 30 min incubation period on ice, cellswere washed twice with FACS™ buffer. It was possible to store the cellsin 500 μL FACS™ buffer at 4° C. up to 7 days. The pellets were washedwith 500 μL 0.1 Saponin in FACS™ buffer once. Subsequently, 50 μL of0.25% Saponin in FACS™ buffer (permeabilisation medium) and 5 μL of thetyrosinase-specific primary antibody were added to the pellet and mixedgently. After a 1 h incubation at room temperature, cells were washedonce with 0.1% Saponin in FACS™ buffer and then 0.5 μL of theCy5-conjugated secondary antibody specific for the primary antibody (1/100 dilution) in 50 μl 0.25% Saponin in FACS™ buffer were added to thepellet and mixed gently. After a 30 min incubation at room temperaturein the dark, the cells were washed with 500 μL 0.1% Saponin in FACS™buffer. Finally, pelleted cells were resuspended in 200 μL of 0.1%Saponin in FACS™ buffer and flow cytometric analysis was performed.

Tetramer Staining and Tetramer Off-Rate

In order to assess the avidity of HLA-A2-restricted tyrosinase-specificCD8⁺ T cells and to perform a sorting with the MOFlo™ High-PerformanceCell Sorter (Dako, Fort Collins, Colo., USA) these T cells were stainedwith a PE-labeled A*0201/htyr369-377/Hβ₂m tetramer (provided by Prof. D.Busch). The cells were washed twice. Each washing step includedresuspension of cells in cold PBS+0.5% human serum, centrifugation at300×g for 5 min and discarding of the supernatant. The incubation volumedepends on the cell number. Up to 10⁶ of cells were incubated in 50 μL25 min with PE-labeled tetramer on ice in the dark ( 1/25 dilution, 2 μLtetramer in 50 μL PBS+0.5% human serum). For the sorting ofHLA-A2-restricted tyrosinase-specific CD8⁺ T cells up to 5×10⁶ cellswere incubated with 6 μL A2/peptide tetramer in 100 μL PBS+0.5% humanserum. Then for an additional 25 min in a ratio of 1/50 CD8-APC antibody(BD Pharmingen, Franklin Lakes, USA) was added. After the staining thecells were washed twice and finally either fixed with 1% paraformaldehyde in FACS™ buffer and analysed by flow cytometry FACSCalibur(Becton Dickinson, Mountain View, Calif.) or diluted in PBS+0.5% humanserum for sorting of the tetramer⁺CD8⁺ T cells in the MoFlo™High-Performance Cell Sorter (Dako, Fort Collins, Colo., USA).

For tetramer off-rate assays, after washing the rebinding of detachedtetramer was prevented by the incubation with saturating amounts ofhybridoma supernatant HB82 (ATCC, Bethesda Md.), which binds HLA-A2independently of the complexed peptide. After increasing time intervalssamples were taken, fixed and analysed by flow cytometry for theintensity of tetramer staining.

Production of Single-Species cRNA

Production of single-species in vitro transcribed RNA, designated hereas cRNA, included four steps: linearisation of the plasmid containing acDNA insert, in vitro transcription based on the promoter sequence andthe cDNA template in the linearised plasmid, polyadenylation ofsynthesised cRNA and cRNA purification. Since substantial amounts of thecDNA template, i.e. plasmid DNA (pDNA), were needed for in vitrotranscription reactions, the plasmid had to be amplified in competentbacteria.

Transformation of Competent Bacteria with Plasmid DNA

In order to obtain larger amounts of pDNA that was received from othergroups as a kind gift, competent bacteria had to be transformed with thepDNA in question and expanded. A 50 μL vial of One Shot TOP10F′competent cells was slowly defrosted on melting ice, a small volume ofpDNA was added and mixed with the bacteria by tapping gently. The cellswere incubated on ice for 30 min, then heated at 42° C. for exactly 30sec and finally placed on ice to cool for 2 min. Rich SOC medium,provided together with the competent cells, was added and transformedbacteria were plated onto LB medium agar plates with the appropriateselection antibiotic. Plates were placed into the bacterial incubatorovernight at 37° C. Since the antibiotic zeocin is light sensitive andits activity is inhibited by high salt concentrations, plates forbacteria transformed with pZeoSV2+/huTyr contained low-salt LB agarmedium and were stored in the dark.

Selection and Expansion of Transformed Bacteria

Only bacteria transformed with pDNA, encoding resistance to anantibiotic, were able to grow and form colonies on selection platesdespite the presence of the corresponding antibiotic in the agar medium.After overnight growth, colonies were plucked from the plates usingsterile tooth-picks. Each individual colony was inoculated into 5 mL ofLB medium containing the appropriate antibiotic in a 15 mL Falcon tube.If previously selected and frozen transformed bacteria were to beexpanded, frozen vials were transferred from the −80° C. freezer ontomelting ice. Approximately 5 μL of the partially thawed bacterialsuspension were then transferred into a 15 mL Falcon tube containing 5mL of LB medium with the appropriate antibiotic. The tubes wereincubated overnight (approximately 12 hr) at 37° C. with vigorousshaking at 150 rpm for efficient growth of transformed bacteria.

Freezing of Transformed Bacteria

Bacteria are the least harmed by the freezing process if the freezingmedium contains 15% of glycerin. Therefore, 400 μL of the overnightbacterial suspension were added to 84 μL of autoclaved 87% glycerin,vortexed, quickly frozen in liquid nitrogen and transferred to −80° C.

Plasmid DNA Extraction from Transformed Bacteria

After overnight culture, bacterial suspensions were centrifuged at5300×g and 4° C. for 10 min. Plasmid DNA extraction from bacterial cellswas performed using the QIAwell® 8 Ultra Plasmid Kit, according tomanufacturer's instructions. Briefly, pelleted cells were lysed. Thesalt and pH conditions in the lysates ensured exclusive binding of DNAto the membrane, while degraded RNA and cellular proteins were notretained. The membrane was then washed with a buffer which disrupts anyDNA-protein interactions allowing the removal of any nucleicacid-binding proteins and other residual contaminants from the pDNA.Additional washing steps removed salts and other non-DNA constituents.Purified pDNA was finally eluted in Tris buffer. The yield of extractedpDNA was 4-8 μg per 5 mL of overnight bacterial culture, depending onthe plasmid.

In Vitro Transcription of Single-Species cDNA into cRNA

Each plasmid was linearised with the appropriate restriction enzyme inorder to produce a template suitable for in vitro transcription. Sinceall plasmids contained the T7 promoter at the 5′ end of cDNA encodingthe desired protein, transcription was performed using the T7 RNApolymerase from the mMESSAGE mMACHINE™ T7 kit according tomanufacturer's instructions. Stability of an RNA molecule and itsefficient use as a template for translation into protein depend on thepresence of a cap at its 5′ end and a poly(A) tail at the 3 end. Apoly(A) tail is always part of a mRNA molecule synthesised by a cell.Therefore, the total cellular mRNA amplification procedure, describedabove, included only the addition of a cap analogue in the in vitrotranscription reaction. However, single-species cRNA produced using cDNAin a plasmid may or may not contain a poly(A) tail, depending on whetherthe tail is encoded in the construct or not. In addition to integratinga cap analogue at the 5′ end in the in vitro transcription reaction,tyrosinase, HLA-A2 cRNAs were polyadenylated at the 3′ end using thePoly(A) Tailing Kit. Both reactions were performed under conditionssuggested by the manufacturer. After in vitro transcription and cRNApurification, the measured yield of cRNA was 60-70 μg per 100 μL of thepolyadenylation reaction mixture.

Purification of cRNA

Purification of cRNA, obtained in in vitro transcription reactions usingas a template either amplified total cellular cDNA or single-speciescDNA in a plasmid, was performed using the RNeasy® Mini Kit according tomanufacturer's instructions. For purification, the procedure startedwith the transfer of the in vitro transcription reaction mixture onto anRNeasy® column. RNA was eluted in DEPC water and aliquots were stored at−80° C.

RNA Transfection into DCs

The cellular plasma membrane serves the vital function of separating themolecular contents of the cell from its external environment. Themembranes are largely composed of amphiphilic lipids which self-assembleinto a highly insulating bilayer. On their quest to capture as muchantigen as possible, immature DCs are well known to take up variousstructures (ranging in size from small molecules to apoptotic bodies)from their surroundings, including RNA. They do so throughmacropinocytosis and endocytosis. Even though simple DC co-incubationwith RNA has been shown to achieve T-cell priming, more aggressivemethods such as lipofection and electroporation proved to be bettertransfection methods.

Electroporation

Electroporation induces reversible permeability of plasma membranes whencells are exposed to short pulses of strong external electric fields.The formation of hydrophilic pores is a result of reorientation oflipids in the bilayer membrane. The molecular mechanism of thisphenomenon is still not well understood. The number of pores and theirdiameter increases with the product of the pulse amplitude and the pulseduration. Whereas small molecules simply diffuse through the pores intothe cytoplasm, larger molecules, like nucleic acids, are driven into thecell by electrophoretic forces. It has also been shown that the presenceof nucleic acids facilitates pore formation. Pores appear within amicrosecond of exposure to the electric field, but it takes minutes forthem to reseal.

First, 2−3×10⁶ DCs or K562 cells were resuspended in at least 170 μLOptiMEM I medium, were put into a 0.4 cm electroporation cuvette andincubated on ice for approximately 3 min. Cooling the suspension beforeelectroporation was advantageous because it slowed down the cellularmetabolism. Thereby, overheating due to electricity was prevented.Furthermore, damage caused by the electric shock and by moleculesreleased from dead cells was minimised. Subsequently, RNA, resuspendedin no more than 100 μL of DEPC water was added, giving a totalelectroporation suspension volume of 270 μL. The suspension was shortlymixed by pipetting and then quickly electroporated. Electroporation wasperformed with 250 V and 150 μF. Immediately after electroporation,cells were transferred into the dendritic cell medium supplemented with2% autologous plasma, 10 mM HEPES, 800 U/mL GM-CSF and 800 U/mL IL-4.The cells were counted and the suspension was quickly aliquoted foreither FACS™ analyses, RNA isolation or functional assays and placedinto the incubator at 37° C. and 5% CO₂.

Functional Assay

In the functional assay, RNA-transfected DCs or K562 cells,peptide-pulsed DCs or tumour cells served as stimulators.Antigen-specific CTL clones served as effectors. In order to measure thestimulatory capacities of stimulators, DCs or other cells were firstco-incubated with the T cells, which were harvested and used inco-incubation cultures 8-14 days after thawing and restimulation. Theamount of IFN-γ secreted by activated T cells was measured in theenzyme-linked immunosorbent assay (ELISA).

Peptide-Pulsed T2 Cells Co-Incubation with T Cells

For co-incubation of T2 cells with CTLs, lower cell numbers were taken.For the exogenous peptide pulsing, 1×10⁶ T2 cells were incubated with 10μg/mL YMD tyrosinase peptide (YMDGTMSQV, Gene Center, LMU, Munich) and10 μg/mL human β₂-Microglobulin (Calbiochem, San Diego, United States).Every 20 minutes the cell suspensions were shaken. After a 2 hincubation at 37° C. and 5% CO₂, the cell suspension was washed once toremove unbound peptide. 100 μL of the peptide-pulsed T2 suspension(1×10⁴ cells in RPMI 1640 supplemented with 12% FCS) was co-incubatedwith 100 μL of the CTL suspension (2×10³ cells in CTL mediumsupplemented with 15% human serum), giving a 5:1 stimulator to effectorratio.

After a 24 h co-incubation of effectors and stimulators, 150 μL of eachsupernatant was harvested and stored at −20° C. The supernatant wasanalysed in the enzyme-linked immunosorbent assay (ELISA).

DC or K562 Co-Incubation with T Cells

Either 12 hr or 24 hr after electroporation, 100 μL of the T cellsuspension (2×10⁴ cells in co-incubation medium) were added to 100 μL ofthe transfected-cell suspension (2×10⁴ or 4×10⁴ cells in dendritic cellmedium supplemented with 2% autologous plasma and 800 U/mL GM-CSF and800 U/mL IL-4), giving a total of 200 μL per well of a 96-well plate and1:4, 1:2, 1:1, 2:1 or 4:1 stimulator to effector ratios.

Untransfected DCs were exogenously pulsed with peptides by adding 10 μLof the peptide solution (100 μL/mL) to 100 μL of the DC suspension(4×10⁴ cells in dendritic cell medium supplemented with 2% autologousplasma and 800 U/mL GM-CSF and 800 U/mL IL-4). After a 2 hr incubationat 37° C. and 5% CO₂, 100 μL, of the CTL suspension (2×10⁴ cells in CTLco-incubation medium) were added, giving a 2:1 stimulator to effectorratio. After a 24 hr co-incubation of effectors and stimulators, 150 μLof each supernatant was harvested and stored at −20° C.

Interferon-Gamma (IFN-γ) ELISA

The measurement of IFN-γ in the co-culture supernatants was performed inELISA using the OptEIA™ Human IFN-γ Set according to manufacturer'sinstructions. Briefly, the ELISA plates were coated with a mouseanti-IFN-γ capture antibody and then blocked with an FBS-containingsolution. In some cases, supernatants were diluted 1:2.5 in theco-incubation medium. The same medium was used to make serial dilutionsof the IFN-γ standard. After IFN-γ from cell culture supernatants orstandard solutions bound to the capture antibodies in the plates, thebiotinylated mouse anti-IFN-γ detection antibody was added together withavidin conjugated to horseradish-peroxidase. To visualise the complexesconsisting of capture antibody, IFN-γ, detection antibody, biotin,avidin and horseradish-peroxidase, H₂O₂ (a substrate for peroxidase) incombination with tetramethylbenzidine (substrate reagents A and B mixed)was added, changing the solution colour into blue. The enzymaticreaction was stopped with 1 M ortho-phosphoric acid. Thereby, the colourof the solution turned yellow, its intensity being directly proportionalto the amount of substrate processed, i.e. indirectly proportional tothe amount of IFN-γ captured. Light absorption in the reaction solutionwas measured at 450 nm. Unknown IFN-γ concentrations were calculatedwith the help of a standard curve which was drawn based on knownconcentration of the IFN-γ standard and corresponding measuredabsorbances.

Cytotoxicity Assay

Cytolytic activity of the tetramer⁺CD8⁺ sorted bulk cell line and T cellclones respectively was analysed in a standard 4 h chromium releaseassay. The two HLA-matched melanoma cell lines MeI A375(HLA-A2-positive, tyrosinase-negative) and MeI IL-2 (HLA-A2-positive,tyrosinase-positive) were used. Briefly, 10⁶ target melanoma cells werelabeled with 200 μCi ⁵¹Cr for 1-1.5 h. ⁵¹Cr-labeled target cells werecultured with T cells in 100 μL/well RPMI 1640 with 12% FCS in V-bottom96-well tissue culture plates (Greiner, Solingen, Germany). To evaluatethe efficacy of CTL-mediated lysis, T cells were serially diluted andthen cocultured with 10³ target cells/well to provide graded effectorcell to target cell (E:T) ratios. After 4 h coculture at 37° C., 50 μLof supernatant were collected and radioactivity was measured in a gammacounter. The percentage of specific lysis was calculated as:100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release). Spontaneous release was assessed byincubating target cells in the absence of effector cells and generallyless than 14%.

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Redirecting human T    lymphocytes towards renal cell carcinoma-specificity by retroviral    transfer of T cell receptor genes. Human Gene Ther., 16(7):79.9-810-   Falk, C. S, and Schendel, D. J. (2002). Allogeneic MHC class I    ligands and their role in positive and negative regulation of human    cytotoxic effector cells. Human Immunol. 63:8-19.-   Gao, L., Bellantuono, I., Elsasser, A., Marley, S. B., Gordon, M.    Y., Goldmann, J. M., Stauss, H. J. (2000). Selective elimination of    leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific    for WT1. Blood 95: 2198-2203.-   Gao, L., Yang, T. H., Tourdot, S., Sadovnikova, E., Hasserjian, R.    and Stauss H. J. (1999). Allo-major-histocompatibility complex    restricted cytotoxic T lymphocytes engraft in bone marrow transplant    recipients without causing graft-versus-host disease. Blood 94:    2999-3006.-   Geiger, C., Regn, S., Noessner, E., Wilde, S., Frankenberger, B.,    Papier, B., Pohla, H., and D. J. Schendel. 2005. Development of a    generic RNA-pulsed dendritic cell vaccine for renal cell    carcinoma, J. Translational Medicine in press.-   Gilboa-E and J. Viewig. 2004. Cancer immunotherapy with    mRNA-transfected dendritic cells. Immunol. Rev. 199:251-63.-   Kolb, H. J., Schmid, C., Barrett, A. J. and Schendel, D. J. (2004).    Graft-versus-leukemia reactions in allogeneic chimeras. Blood    103:767-776.-   Kolb, H. J., Schattenberg, A., Goldman, J. M., Hertenstein, B.,    Jacobsen, H., Arcese W., Ljungman, P., Ferrant, A., Verdonck, L.    Niederwieser, B. et al. 1995. Graft-versus-leukemia effect of donor    lymphocyte transfusions in marrow grafted patients. Blood 86:2041.-   Levings, M. K. and M. G. Roncarolo. 2005. Phenotypic and functional    differences between human CD4+CD25+ and type 1 regulatory T cells.    Curr. Top. Microbiol. Immunol. 293:303-326.-   Liao, X., Yongging, L., Bonini, C., Nair, S., Gilboa, E.,    Greenberg, P. D., Yee, C. (2004). 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FURTHER REFERENCES

-   Belinda Palermo, Silvia Garbelli, Stefania Mantovani, Elisabetta    Scoccia, Gian Antonio Da Prada, Paola Bernabei, M. Antonietta    Avanzini, Valeria Brazzelli, Giovanni Borroni and Claudia Giachino    (2005). Qualitative difference between the cytotoxic T lymphocyte    responses to melanocyte antigens in melanoma and vitiligo. Eur. J.    Immunol. 35: 3153-3162.-   Cassian Yee, Peter A. Savage, Peter P. Lee, Mark M. Davis, and    Philip D. Greenberg (1999). Isolation of High Avidity    Melanoma-Reactive CTL from Heterogeneous Populations Using    Peptide-MHC Tetramers. The Journal of Immunology: 2227-2234.-   Matthias Wölfl, Stefan Schalk, Martin Hellmich, Katharina M. Huster,    Dirk H. Busch, and Frank Berthold (2004). Quantitation of MHC    Tetramer-Positive Cells From Whole Blood: Evaluation of a    Single-Platform, Six-Parameter Flow Cytometric Method. Wiley-Liss,    Inc. Cytometry Part A 57A:120-130.

1-18. (canceled)
 19. An isolated nucleic acid coding for a T cellreceptor (TCR), wherein the isolated nucleic acid coding for thetransgenic TCR is obtainable by a method comprising: (a) providing anucleic acid encoding a patient-derived major histocompatibility complex(MHC) molecule and an antigen or a second nucleic acid encoding theantigen; (b) co-transfecting or introducing both compounds as defined in(a) into antigen presenting cells (APCs) derived from a healthy donor;(c) priming peripheral blood lymphocytes (PBLs) derived from the healthydonor with the APCs; (d) selecting a T cell that is specific for theMHC-antigen ligand; and (e) cloning the TCR of the selected T cell,whereby an isolated nucleic acid coding for a TCR is obtained.
 20. Avector, which comprises the nucleic acid of claim
 19. 21. The vector ofclaim 20, which is a plasmid or a retroviral vector.
 22. A PBMC, whichhas been transformed with the vector of claim
 20. 23. A pharmaceuticalcomposition which comprises the PBMC of claim 22 and a pharmaceuticallyacceptable carrier.
 24. The pharmaceutical composition of claim 23,which is an infusion, injection or a vaccine. 25-26. (canceled)
 27. Thenucleic acid of claim 19, wherein the APCs are dendritic cells (DCs).28. A PBMC, which has been transformed with the vector of claim 21.